JP2791303B2 - Identification method for identifying the number of colonies indicated by one connection region, and colony counting device using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a colony counting device for counting the number of colonies generated in a culture medium, for example, a mutagenicity test using a microorganism (or
(A reverse mutation test using bacteria).

[0002]

2. Description of the Related Art A mutagenicity test using a microorganism (or a reverse mutation test using a bacterium) is performed as a test for determining whether or not a drug has carcinogenicity. As one of them, a test called Ames test is performed.

Here, the Ames test will be described. As shown in FIG. 8, agar (colorless and transparent) 2 as a medium is poured into a 90 mm-diameter dish 1 together with 10 ⁹ or more predetermined bacteria which are easily mutated and solidified (hereinafter, this is referred to as “ Plate "). FIG. 8 is a view showing the plate 3, FIG. 8 (a) is a plan view thereof, and FIG. 8 (b) is a sectional view thereof. Then, a chemical to be tested is applied to the surface of the agar 2 and the plate is kept at a predetermined temperature (37 ° C.) for a predetermined time (48 hours). Thereafter, the number of colonies 4 generated on the agar 2 is counted. I do. The final conclusion of this test is that there is a positive (carcinogenic) when the number of colonies with respect to the change in the concentration of the test substance applied is more than twice the number of colonies when nothing is applied. Is done. This test is called the Ames test.

[0004] In addition to the Ames test, the number of colonies generated in the medium is counted in other mutagenicity tests using microorganisms and reversion tests using bacteria.
A test result is obtained based on the counted value, and it is extremely important to accurately count the number of colonies generated in the medium. However, due to the nature of the test, the number of colonies is twice as high when the test substance is not applied,
The absolute value itself of the number of colonies does not matter, and the counting condition only needs to be accurately defined in order to increase the reliability of the relative value of twice.

However, a conventional colony counting apparatus for counting colonies generated in a culture medium employs a so-called line scanning method, in which the culture medium is scanned linearly or spirally, and the number of times the scanning line passes over the colony is determined. Since the number of colonies was counted based on the number of colonies, the number of colonies could not be counted accurately. That is, in the line scanning method, when the colony 4 has a somewhat flat circular shape, the colony 4 is recognized if the colony 4 projects widely in the scanning direction of the scanning line 10 (FIG. 9A). If the projection is narrow, the colony 4 cannot be recognized (see FIG. 9).
(B)). Also, if colonies 4 overlap,
Depending on the direction of the scanning line 10 with respect to the colony 4, two colonies 4 are recognized (FIG. 9C), or only one colony 4 is recognized (FIG. 9D). As described above, since the recognition of the colony 4 changes depending on the direction of the scanning line 10 with respect to the colony 4, the count value of the colony 4 changes depending on the direction in which the plate is placed. I ca n’t,
The error in the count value of the colony 4 was, for example, 20% or more. FIG. 9 is a diagram schematically illustrating the state of the line scan.

Therefore, the fact is that a person looks at the colony 4 and counts the number. Therefore, counting the colonies 4 requires a great deal of labor. In addition, when a human counts the number of colonies 4, a large colony 4 and a small colony 4 are mixed (for example, 7 to 8 mm).
If the colonies 4 having a size of about 500 μm are mixed with the colonies 4 having a size of about 4, the small colonies 4 are easily overlooked. That is, the condition of the count is changed by the filter based on the familiarity of the human eyes, and the colony 4 cannot be counted accurately.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above circumstances, and a colony counting apparatus capable of counting the number of colonies with high accuracy, and one connecting apparatus which can be used for this. An object of the present invention is to provide an identification method for identifying the number of colonies indicated by an area.

[0008]

The present inventors have observed the colonies in detail and found that (1) one colony is almost circular, (2) the colony itself is cloudy white, (3) Because the center of the colony is thick,
The concentration increases as it approaches the center, and the concentration gradient is relatively gentle. (4) The size of the colony cannot be specified. (A colony can grow more if it can absorb more nutrients, but a large number of colonies are generated.) If so, competition for nutrient uptake will occur and its growth will be poor and small.
Conversely, when only a few colonies are generated, the colonies grow well and may have a diameter of about 7 to 8 mm. ) And other characteristics of the colony.

The present invention has been made based on such findings.

To solve the above-mentioned problems, a colony counting device according to a first aspect of the present invention is a colony counting device for counting the number of colonies generated in a culture medium, comprising: Means for obtaining a connected region corresponding to a colony based on image data obtained from the imaging means, and for each obtained connected region, a circumscribed rectangle circumscribing the connected region and having a longest longest side A means for calculating the ratio between the long side and the short side of the circumscribed rectangle, and a weight coefficient that is a weight coefficient that is predetermined according to the ratio and that corresponds to the ratio obtained for each of the connected regions. And means for integrating. In addition, the expression form of the connection region corresponding to the colony may be a form of a contour line or a form of a surface.

In a colony counting apparatus according to a second aspect of the present invention, there is provided a colony counting apparatus for counting the number of colonies generated in a culture medium. Means for determining a connected region corresponding to the colony based on the image data;
For each of the obtained connected regions, a normal to the tangent at each point on the outline of the connected region is obtained, and the number of locations where the intersections are concentrated is obtained according to the distribution of the intersections of the normals. Means, and means for integrating the number obtained for each of the connected regions. In addition, the expression form of the connection region corresponding to the colony may be a form of a contour line or a form of a surface.

[0012] In a colony counting apparatus according to a third aspect of the present invention, in the colony counting apparatus according to the first or second aspect, the means for obtaining a connected region corresponding to a colony includes an image obtained from the imaging means. The method includes means for determining a connected region having a concentration corresponding to a colony based on the data, wherein the connected region having a concentration corresponding to the colony is a connected region corresponding to the colony.

In a colony counting apparatus according to a fourth aspect of the present invention, in the colony counting apparatus according to the first or second aspect, the means for obtaining a connection region corresponding to a colony is an image obtained from the imaging means. Means for determining a connected region having a concentration corresponding to the colony based on the data, and means for determining the size of the connected region having a concentration corresponding to the colony, wherein a predetermined portion of the connected regions having a concentration corresponding to the colony is included. The connecting region having a size equal to or larger than the size of the colony is a connecting region corresponding to the colony.

In a colony counting apparatus according to a fifth aspect of the present invention, in the colony counting apparatus according to the first or second aspect, the means for obtaining a connected region corresponding to a colony is an image obtained from the imaging means. Including extraction means for extracting a contour line corresponding to the colony based on the data,
The extracting means obtains the position of the peak value of the density gradient where the peak value falls within a predetermined range, and obtains the contour based on the position.

In a colony counting apparatus according to a sixth aspect of the present invention, in the colony counting apparatus according to any one of the first to fifth aspects, the means for obtaining a connection region corresponding to a colony is obtained from the imaging means. Means for masking an area outside the colony counting range based on the obtained image data.

A colony counting device according to a seventh aspect of the present invention is the colony counting device according to any one of the first to sixth aspects, wherein the imaging means has a one-dimensional image sensor. Is a scanner for scanning.

The colony counting apparatus according to an eighth aspect of the present invention is the colony counting apparatus according to any one of the first to seventh aspects, wherein the colony counting apparatus is obtained by the integrating means with the image data obtained from the imaging means. A storage unit for storing the integrated values in association with each other. In addition, without being limited to the colony counting device according to any one of the first to seventh aspects, in a colony counting device that counts the number of colonies generated in the culture medium, an imaging unit that images the culture medium in which the colonies have occurred, Means for counting colonies based on the image data obtained from the imaging means, and means for storing the count value of the obtained colonies and the image data obtained from the aforementioned imaging means in association with each other, A colony counting device can also be provided.

In the identification method according to a ninth aspect of the present invention, the medium in which a colony has occurred is imaged by an imaging means, and a connected region corresponding to the colony is determined based on image data obtained from the imaging means. A method for identifying the number of colonies indicated by one of the connected regions, wherein the obtained one connected region is a circumscribed rectangle circumscribing the connected region, and the circumscribed rectangle having the longest longest side. The ratio between the long side and the short side is obtained, and the connection area indicates a weight coefficient that is predetermined according to the ratio and corresponds to the ratio obtained for the connection area. It is the number of colonies.

[0019] The identification method according to the tenth aspect of the present invention comprises:
An identification method for capturing an image of a medium in which a colony has occurred by an imaging unit, determining a connected region corresponding to the colony based on image data obtained from the imaging unit, and identifying the number of colonies indicated by one of the obtained connected regions In the obtained one connected region, a normal to a tangent at each point on the outline of the connected region is obtained, and the intersections are concentrated according to the distribution of the intersections of the normals. The number of locations where the connection area exists is determined, and the number is used as the number of colonies indicated by the connection region.

According to the first and eighth aspects, the culture medium in which the colony has occurred is imaged by the imaging means, and a connection region corresponding to the colony is obtained based on the image data obtained from the imaging means. Then, the ratio of the long side to the short side of the circumscribed rectangle circumscribing the connected area and having the longest side is determined for the obtained connected area. A weighting factor that is predetermined according to the ratio and that corresponds to the ratio obtained for the connected region is identified as the number of colonies indicated by the connected region. Since the number of colonies indicated by the connected region is identified in accordance with the ratio in this manner, since one colony is substantially circular, by appropriately setting the weighting factor in accordance with the ratio, In consideration of the overlap of colonies, the number of colonies indicated by the connection region can be identified with sufficiently high accuracy. Since the ratio of the long side to the short side of the circumscribed rectangle in which the long side of the connected region obtained from the image data which is the two-dimensional data is the longest is used as a reference for identifying the number of colonies, the direction of the plate, etc. Without any influence, the number of colonies indicated by the connection region can be identified. In the first aspect, the number of such colonies is identified for each connected region, and the number (weight coefficient) is integrated,
The total number of colonies is obtained. As described above, the number of colonies indicated by one connection region can be accurately identified, so that the total number of colonies can be obtained with high accuracy.

According to the second and ninth aspects,
The culture medium in which the colony has occurred is imaged by the imaging means, and a connection region corresponding to the colony is determined based on the image data obtained from the imaging means. Then, a normal to the tangent at each point on the outline of the connected region is obtained for the obtained connected region, and the number of locations where the intersections are concentrated is determined according to the distribution of the intersections of the normals. Is determined, and the number is identified as the number of colonies indicated by the connection region. As described above, the number of colonies indicated by the connection area is identified by the number of locations where the intersections are concentrated according to the distribution state of the intersections of the normals. Are concentrated near the center of the colony, so that the number of colonies indicated by the connection region can be identified with sufficiently high accuracy in consideration of colony overlap. Then, according to the distribution of the intersection of the normal to the tangent at each point on the outline of the connected region obtained from the image data which is two-dimensional data, the number of locations where the intersections are concentrated is used to identify the number of colonies. Since it is used as a reference, the number of colonies indicated by the connection region can be identified without any influence on the direction of the plate or the like. In the second aspect, the number of such colonies is identified for each connection region, and the numbers are integrated to obtain the total number of colonies. As described above, the number of colonies indicated by one connection region can be accurately identified, so that the total number of colonies can be obtained with high accuracy.

In the first, second, eighth, and ninth aspects, the method for obtaining the connection region corresponding to the colony is not particularly limited. For example, as in the third aspect, May be determined, and this connected region may be used as the connected region corresponding to the colony (the connected region to be counted). Further, as in the fourth aspect, a connected region having a concentration corresponding to a colony is determined, and a connected region having a size equal to or larger than a predetermined size among the connected regions is connected to a target to be subjected to a counting process. It may be an area. Conventionally, when a human counts the number of colonies, the number of colonies smaller than a certain size is not counted, but is not counted depending on the ambiguous judgment by the human. In this regard, in the case of the fourth aspect, the counting is not performed depending on the determination of the ambiguous size by a human, but rather is not accurately counted. The number of colonies can be counted with high accuracy.

Further, as in the fifth aspect, a connected region corresponding to a colony may be obtained by extracting a contour line corresponding to the colony. In this case, since the concentration gradient of the foreign substance is usually steep while the concentration gradient of the colony is relatively gentle, the position of the peak value of the concentration gradient and the peak value If a position within a predetermined range is obtained and the outline is obtained based on the position, even if a foreign substance is mixed, the foreign substance is not counted and the colony is more accurately detected. Can be counted.

Further, as in the sixth aspect, the means for obtaining a connected area corresponding to a colony includes means for masking an area outside the colony counting range based on image data obtained from the imaging means. You may have. If the position where the plate is to be placed is determined in advance and the plate is accurately placed at that position, or if a mask for optically masking the edge of the petri dish is provided in advance, based on the image data as in the sixth aspect, No masking means is required. However, it is preferable to employ a masking means based on image data as in the sixth aspect, since it is not necessary to accurately place a plate at a predetermined position or to facilitate or install an optical mask.

As the image pickup means, any image pickup device such as a two-dimensional CCD camera can be adopted. As in the seventh embodiment, a one-dimensional image sensor having a one-dimensional image sensor is used.
If a scanner that scans a three-dimensional image sensor is used,
Since the resolution of the one-dimensional image sensor is higher than that of the one-dimensional image sensor, high-resolution image data can be obtained, and colonies can be counted with higher accuracy, which is preferable.

Incidentally, the condition (number, size, etc.) of the colonies generated in the medium changes according to the elapsed time. Therefore, in the aforementioned Ames test, the elapsed time and the like are strictly specified. Therefore, it is impossible to preserve the plate in which the colonies have been counted once as it is. For this reason, when the number of colonies is counted using the conventional colony counting device or when a human counts the number of colonies, if doubts arise in the test results later, the doubts are confirmed. It is impossible to confirm the reliability of the test.

In this regard, according to the colony counting apparatus according to the eighth aspect, the image data obtained from the imaging means and the integrated value (colonized count value) obtained by the integrating means are stored in association with each other. In the case where doubts arise in the test result later, the doubt can be resolved by comparing the colony count value with the image based on the corresponding image data, and the test can be performed. Reliability can be improved.

[0028]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a colony counting device according to a first embodiment of the present invention will be described with reference to FIGS.

FIG. 1 is a block diagram showing a colony counting device according to the present embodiment. FIG. 2 is a flowchart illustrating an example of the operation of the processing unit 22. FIG. 3 is an explanatory diagram showing predetermined processing contents of the processing unit 22.

The colony counting device according to the present embodiment
An imaging device 21 for imaging the plate 3 shown in FIG.
U, a processing unit 22 including a signal processing circuit, an input device such as a keyboard and a mouse for giving various instructions and the like to the processing unit 22, an image data from the imaging device 21 and the processing unit 22.
Memory 24 for storing image data obtained in the calculation process by the computer, and memory 2 for storing the operation program of the processing unit 22, the data obtained in the calculation process, and the colony count value.
5, a display device 26 such as a CRT for displaying a colony count value and the like, and an image data from the imaging device 21 and an obtained colony count value, which are not shown in the drawing, are stored in association with each other. And an external storage device such as a floppy disk device or a magneto-optical disk device.

In the present embodiment, a scanner having a one-dimensional image sensor such as a one-dimensional CCD and scanning the one-dimensional image sensor is used as the imaging device 21. One-dimensional image sensors are preferred because of their high resolution. However, any imaging device such as a two-dimensional CCD camera may be adopted as the imaging device 21.

Further, the light source may be arranged below the plate 3 so that the image of the plate by the transmitted light is picked up by the image pickup device 21, or the light source is arranged above the plate 3 and the plate The image may be captured by the imaging device 21.

Next, an example of the operation of the processing section 22 in the present embodiment will be described with reference to FIG.

When the processing section 22 starts the operation, first,
In step S1, initialization such as setting the colony count value in the memory 25 to zero is performed. Note that, as optical adjustment, contrast and brightness settings and image contour adjustment are performed in advance so that the density difference between the colony 4 and the agar 2 of the image captured from the imaging device 21 becomes clear.

Next, the processing section 22 fetches the image data from the imaging device 21 and stores it in the image memory 24 in step S2. In the present embodiment, the image data is data of 256 gradations in which the pixel density is represented by 0 to 255, but it is needless to say that the image data is not limited to this.

Thereafter, in step S3, the processing unit 22 determines the area outside the counting range of the colony 4 (in this example, the edge portion of the petri dish 1 and the outer area thereof) based on the image data taken into the image memory 24. Mask it. That is, of the image data, the density of the region outside the counting range in the captured image data is set to “0”, and the counting range (in this example, the region of the agar 2 surrounded by the edge of the petri dish 1) The density of the image data is kept in the captured state.

The counting range is identified, for example, as follows. The density of the area outside the edge of the Petri dish 1 is different from the density of the edge of the Petri dish 1, the density of the agar 2 and the density of the colony 4, and is usually almost 0 (the highest density). The concentration of colony 4 is lower than the concentration of agar 2, and the concentrations of both are different. The range of the concentration of the edge portion of the petri dish 1 is wide, including the range of the concentration of the colony 4 and the range of the concentration of the agar 2. Since the shape of the petri dish 1 is circular, the counting range is also concentric and circular. In order to know the counting range, it is necessary to find its center and radius. If the size of the petri dish 1 is known, it is only necessary to find the center. The center of the counting range is obtained as follows. From one side in the X direction, the densities of pixels in a row arranged in the Y direction (a direction orthogonal to the X direction) are sequentially examined, and the position of the pixel with the lowest density found first is considered to be the outer edge of the petri dish 1. The X coordinate of the pixel is the X coordinate of the center of the counting range. Similarly, from one side in the Y direction, the densities of the pixels in the row arranged in the X direction are sequentially examined, and the position of the pixel with the lowest density found first can be considered as the outer edge of the petri dish 1. The Y coordinate of the pixel becomes the Y coordinate of the center of the counting range. Thus, the center coordinates of the counting range are obtained. If the thickness of the edge portion of the petri dish 1 is known, the radius of the counting range can be determined from the center coordinates and the coordinates of the first found low density pixel. on the other hand,
If the thickness of the edge of the Petri dish 1 is not known, the observation is continued by continuing the observation from the position where the outer edge of the Petri dish 1 is first found, and finding the pixel having the density corresponding to the agar 2 as described above. The radius of the counting range is determined from the coordinates of the pixel and the center coordinates of the counting range. In this way, the counting range can be identified.

Next, in step S4, the processing unit 22 searches for a pixel in a predetermined density range corresponding to the colony 4 based on the masked image data, and sets the same density range in eight directions around the pixel. One connected area (island), which is an area composed of adjacent pixels in a predetermined density range corresponding to the colony 4, is determined while checking whether there is any pixel included. Of course, an isolated single pixel in a predetermined density range corresponding to the colony 4 is also one connected region. Similarly,
All connected regions within a predetermined density range are obtained.

Next, in step S5, the processing unit 22 determines the area of each connected region determined in step S4 (for example,
(The number of pixels forming the connection region).

Thereafter, in step S6, as shown in FIG. 3, the processing unit 22 forms a circumscribed rectangle circumscribing the connection region with respect to each connection region obtained in step S4 and has the longest side. Find the circumscribed rectangle. FIG. 3 shows one connection region 31 and a circumscribed rectangle 32 as described above for one connection region 31 in order to simplify the drawing.

Next, in step S7, the processing unit 22 assigns a number to each of the connected regions obtained in step S4 in ascending order of area.

Next, the processing unit 22 selects the leading region of the connected region in step S8, and determines in step S9 whether the area of the selected connected region is equal to or larger than a predetermined value. If the area of the selected connected region is smaller than the predetermined value, the processing unit 22 ends the operation. On the other hand, if the area of the selected connected region is equal to or larger than the predetermined value, the process proceeds to step S1
Move to 0. In the present embodiment, the area is used as the size of the connection region, but, for example, the length of the short side of the circumscribed rectangle with respect to the connection region may be used instead.

In step S10, the processing section 22 calculates the ratio (l / m) between the long side 1 and the short side m of the circumscribed rectangle of the selected connected area.

Next, in step S11, the processing unit 22 determines that the ratio calculated in step S10 is the first range (the range in which the selected connected region is highly likely to be one colony, for example, , 1.0-1.2
Is determined. If the ratio calculated in step S10 falls within the first range, a weighting factor “1” is added to the colony count value in the memory 25 in step S12, and if not, the process proceeds to step S13.

In step S13, the processing section 22
If the ratio calculated in step S10 is in the second range (the selected connected region is a highly probable range in which two colonies overlap, for example, 1.2 to
2.0 range). Step S1
If the ratio calculated in 0 falls within the second range, step S
At 14, the colony count value in the memory 25 is assigned a weighting factor of “2”.
Otherwise, the process proceeds to step S15 without adding to the colony count value. That is, when the ratio is larger than 2.0, this is equivalent to adding the weighting factor “0”.

In step S15, the processing section 22
It is determined whether or not the number of the selected connection area is the last number assigned in step S7. If it is the last number,
The processing unit 22 ends the operation. On the other hand, if it is not the last number, in step S16, the processing unit 22 selects the next numbered connected area, returns to step S9, and repeats steps S9 to S16. The colony count value in the memory 25 is displayed on the display device 26.

Although not shown in FIG. 2, the processing unit 2
2 stores the colony count value in the memory 25 and the image data fetched into the image memory 24 in step S2 before terminating the operation, by using the above-mentioned external storage device (exactly, a floppy disk or a magneto-optical disk). Etc.).

According to the present embodiment, the number of colonies indicated by the connection region is identified according to the ratio as described above. Since one colony is substantially circular, the weight coefficient is , The number of colonies indicated by the connected region can be identified with sufficiently high accuracy, taking into account the overlap of colonies. Since the ratio of the long side and the short side of the circumscribed rectangle having the longest side to the longest side of the connected area obtained from the image data as the two-dimensional data is used as the reference for the number of colonies, the direction of the plate 3 Is not affected at all,
The number of colonies indicated by the connection region can be identified. In this way, the number (weighting factor) of the number of colonies indicated by the connected region that has been accurately identified is added up, and the total number of colonies is obtained. Therefore, the total number of colonies is also obtained with high accuracy.

By the way, in this embodiment, the ratio 1.0
A weighting factor of 1 is assigned to.
A weighting factor of 2 is assigned to 2 to 2.0, and the ratio mainly corresponds to a single colony and mainly 2
Only those corresponding to heavy colonies were counted, and the connected regions with other ratios were not counted as dust and scratches. However, 90% of the group of connected regions corresponded to colonies that exist alone, and 80-90% of the remaining connected regions corresponded to two colonies overlapping, and the rest (1-2% of the total). %) Is equivalent to a region where three or more colonies overlap, and the probability that three or more colonies overlap is very low. Therefore, the total number of colonies was accurately obtained, and the number of colonies was actually counted with an error of about 3%.

Since the weighting factor can be regarded as an expected value of how many colonies correspond to the connected region of the corresponding ratio, the weighting factor may be determined not only as an integer but also as a decimal. In this case, when the count value of the colony obtained by integrating the weighting factors has a portion below the decimal point, the portion below the decimal point may be rounded off as necessary.

In this embodiment, the colony count value finally obtained in the memory 25 and the image data taken in the image memory 24 in step S2 are stored in a floppy disk or a magnetic recording medium in association with each other. If the test results are questionable later, the doubt can be resolved by comparing the colony count value with the image based on the corresponding image data, and the test reliability should be improved. Can be.

As can be seen from the above description, in the present embodiment, steps S2 to S9 correspond to means for obtaining a connected area corresponding to colony 4. If the position where the plate 3 is to be placed is determined in advance and the plate 3 is accurately placed at that position, or if a mask for optically masking the edge of the petri dish 1 is provided in advance, the mask processing in step S3 is deleted. Is also good. Steps S5 and S
9 may be deleted.

Next, a colony counting apparatus according to a second embodiment of the present invention will be described with reference to FIGS.

FIG. 4 is a flowchart showing an example of the operation of the processing section 22 in the colony counting device according to the present embodiment. FIG. 5 is an explanatory diagram showing predetermined processing contents of the processing unit 22. FIG. 6 is a diagram illustrating another predetermined processing content of the processing unit 22. FIG. 7 is a diagram illustrating still another predetermined processing content of the processing unit 22.

The colony counting device according to the present embodiment has the same basic configuration as the colony counting device according to the first embodiment described above, and FIG. 1 is a block diagram showing the colony counting device according to the present embodiment. is there. The only difference between the two is the operation of the processing unit 22. Therefore, only the operation of the processing unit 22 will be described here.

Steps S21 to S29 in FIG. 4 are the same as steps S1 to S9 in FIG. 2 described above, and a duplicate description thereof will be omitted. FIG. 5A shows one connection region 41 and a circumscribed rectangle 42 corresponding thereto.

If the result of determination in step S29 is that the area of the selected connected region is equal to or larger than a predetermined value, the flow shifts to step S30.

In step S30, the processing unit 22 extracts the contour of the selected connected area. The extraction of the outline can be directly obtained, for example, based on the outline of the selected connected region which is the connected region obtained in step S24. In this case, even if there is a foreign substance mixed in the connected area obtained in step S24 and there is a connected area due to the foreign substance, the outline of the foreign substance is extracted. Occurs. Therefore, in the present embodiment, the processing unit 22 employs a contour line extraction method described below in step S30.

Here, a method of extracting a contour line that can extract the contour line of a connected region by a colony while avoiding the extraction of the contour line of a connected region by a foreign substance will be described. FIG. 6A is a diagram schematically showing an image of a colony, FIG. 6B is a diagram showing the density along the center line in FIG. 6A, and FIG. 6C is a diagram in FIG. FIG. 6D shows the differential value of the density along the center line in FIG. 6D, FIG. 6D shows the density along the center line in FIG. 6A, and FIG. FIG. 6F is a diagram showing a differential value (density gradient) of the density along the center line of FIG. 6, and FIG. 6F is a diagram showing a differential value (density gradient) of the density along the center line in FIG. 6D. As shown in FIGS. 6D and 6E, the concentration gradient of the foreign matter is steep, whereas FIG.
As shown in (b), the concentration gradient of the colony is relatively gentle. Therefore, while the peak value of the concentration gradient of the foreign matter (the value at the top of the mountain and the value at the bottom of the valley) is large as shown in FIG. 6 (f), the concentration gradient of the colony as shown in FIG. The peak values (the values at the top of the mountain and at the bottom of the valley) are relatively small. For this reason, if the peak value of the density gradient does not fall within the predetermined range, it is ignored, and if the peak value of the density gradient falls within this range, but the contour is extracted based on the position of the peak value, it is possible to detect the presence of foreign matter. It is possible to extract the outline of the connected region by the colony without extracting the outline of the connected region. In other words, the position of the peak value of the concentration gradient in the gradient direction, where the peak value falls within a predetermined range, is determined, and the contour is determined based on the position. Can be extracted without extracting the outline of the connected region by the colony. Note that, in the present embodiment, the processing unit 22 performs an operation such as differentiation based on the image data corresponding to the area in the circumscribed rectangle 42 obtained in step S26 in step S30, so that the contour line described above is obtained. An extraction method is realized.

Next, in step S31, as shown in FIG. 5B, the processing unit 22 determines each point of the contour 43 extracted in step S30 (for example, all points of the pixels on the contour 43). , Or each point on the contour line 43 at predetermined intervals) with respect to the tangent line.

Next, in step S32, the processing section 22 determines an intersection of adjacent normals among the normals determined in step S31. However, for example, intersections of normal lines at predetermined intervals may be obtained.

Thereafter, in step S33, the number of locations where the intersections are concentrated is determined according to the distribution of the intersections determined in step S32. An example of the specific method will be described with reference to FIG. However, it is not limited to the example shown in FIG. First, as shown in FIG. 7A, a step S3 is performed for each position in the X′Y ′ coordinate system corresponding to the circumscribed rectangle 42 obtained in the step S26.
The number of intersections obtained in step 2 is arranged. In this example, FIG.
The rectangle 42 'in the middle has the same size and the same number of pixels as the circumscribed rectangle 42 in FIG. In FIG. 7A, the rectangle 42 'is composed of 10.times.15 pixels, and the cells indicated by broken lines represent pixels, and the number written in the pixel is the number of the intersections located at the position of the pixel. Next, a square window (3 × 3 pixels) having a length of a predetermined length of the short side of the circumscribed rectangle 42 (30% of the short side in the example shown in FIG. 7A) as one side length ) 50 is sequentially moved such that its upper left pixel overlaps each pixel in rectangle 42 ',
The number of intersections in the window 50 is sequentially added, and the added value is written to a pixel in the X′Y ′ coordinate system shown in FIG. 7B at a position corresponding to the position of the upper left pixel of the window 50. Note that the rectangle 42 ″ in FIG. 7B also has the same size and the same number of pixels as the circumscribed rectangle 42 in FIG. 5A. In FIG. It is composed of 10 × 15 pixels, and the cells indicated by broken lines represent pixels, and the number written in the pixel is the sum of the number of intersections in the window 50 located at the corresponding position as described above. As shown in FIG. 7B, when the added value is written to all the pixels in the rectangle 42 ″, the total value of the added values written to each pixel (in the example shown in FIG. 7B, "1116")
A value of a predetermined ratio of the total value (3% of the total value “1116”, “33.48” in the example shown in FIG. 7B) is obtained. In the coordinate system of FIG. 7B, a pixel in which an added value larger than this value is written is searched, and a connected region (island) of the pixel is obtained. In the example shown in FIG. 7B, two connected regions surrounded by a thick solid line are obtained. The number of the connection areas (two in FIG. 7B) is the number of places where the intersections are concentrated.

Next, the processing section 22 executes step S in FIG.
At 34, the number of the points where the intersections are concentrated, obtained at step S33, is added to the colony count value in the memory 25, and the process proceeds to step S35.

In step S35, the processing section 22
It is determined whether or not the number of the selected connection area is the last number assigned in step S27. If it is the last number, the processing unit 22 ends the operation. On the other hand, if it is not the last number, in step S36, the processing unit 22 selects the next numbered connection area, returns to step S29, and repeats steps S29 to S36. The colony count value in the memory 25 is displayed on the display device 26.

Although not shown in FIG. 4, the processing unit 2
2 stores the colony count value in the memory 25 and the image data captured in the image memory 24 in step S22 before terminating the operation, by using the above-mentioned external storage device (exactly, a floppy disk or a magneto-optical disk). Etc.).

According to the present embodiment, the number of places where the intersections are concentrated depends on the distribution of the intersections of the normals to the tangents at each point on the contour of the connection area. Since the number of colonies indicated by is identified, one colony is substantially circular and the intersection is concentrated near the center of the colony. Can be identified. And
According to the distribution of intersections of the normals to the tangents at each point on the outline of the connected area obtained from the image data that is two-dimensional data, the number of points where the intersections are concentrated is used as a criterion for identifying the number of colonies. Therefore, the number of colonies indicated by the connection region can be identified without any influence on the direction of the plate or the like. The number (weighting factor) of the number of colonies indicated by the connected region is thus accurately identified.
Are integrated to obtain the total number of colonies, so that the total number of colonies can be obtained with high accuracy.

In the present embodiment, the finally obtained colony count value in the memory 25 and the image data taken into the image memory 24 in step S22 are stored in a floppy disk or a magnetic recording medium in association with each other. If the test results are questionable later, the doubt can be resolved by comparing the colony count value with the image based on the corresponding image data, and the test reliability should be improved. Can be.

As can be seen from the above description, in the present embodiment, steps S22 to S30 correspond to means for obtaining a connected region corresponding to colony 4. If the position where the plate 3 is to be placed is determined in advance and the plate 3 is accurately placed at that position, or if a mask for optically masking the edge of the petri dish 1 is provided in advance, step S
The mask processing of No. 3 may be deleted. Step S2
5, S29 may be deleted.

While the embodiments of the present invention have been described above, the present invention is not limited to these embodiments.

For example, in the flow chart shown in FIG. 2, between step S9 and step S10, step 30 in FIG. The above-described processing for extracting a line may be inserted. In this case, in step S10 in FIG.
The ratio between the long side and the short side of the circumscribed rectangle circumscribing the contour obtained by step 30 and having the longest side may be determined.

[0071]

As described above, according to the present invention,
The number of colonies can be counted accurately.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a colony counting device according to first and second embodiments of the present invention.

FIG. 2 is a flowchart illustrating an example of an operation of a processing unit in the colony counting device according to the first embodiment of the present invention.

FIG. 3 is an explanatory diagram showing predetermined processing contents of a processing unit in the colony counting device according to the first embodiment of the present invention.

FIG. 4 is a flowchart illustrating an example of an operation of a processing unit in the colony counting device according to the second embodiment of the present invention.

FIG. 5 is an explanatory diagram showing predetermined processing contents of a processing unit in a colony counting device according to a second embodiment of the present invention.

FIG. 6 is an explanatory diagram showing another predetermined processing content of the processing unit in the colony counting device according to the second embodiment of the present invention.

FIG. 7 is an explanatory diagram showing still another predetermined processing content of the processing unit in the colony counting device according to the second embodiment of the present invention.

8A and 8B are views showing the plate 3, FIG. 8A is a plan view thereof, and FIG. 8B is a sectional view thereof.

FIG. 9 is a diagram schematically showing a state of a conventional line scan.

[Explanation of symbols]

 Reference Signs List 1 Petri dish 2 Agar (medium) 3 Plate 4 Colony 21 Imaging device 22 Processing unit 23 Input device 24 Image memory 25 Memory 26 Display device

────────────────────────────────────────────────── ⁶ Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI G06T 7/00 G06F 15/70 330R (56) References JP-A-62-251635 (JP, A) JP-A-2-307164 ( JP, A) JP-A-49-70669 (JP, A) JP-A-2-27259 (JP, A) JP-A-1-296974 (JP, A) JP-A-7-147997 (JP, A) International publication 94/26870 (WO, A) (58) Field surveyed (Int. Cl. ⁶ , DB name) C12M 1/34 C12Q 1/06

Claims (10)

(57) [Claims] 1. A colony counting device for counting the number of colonies generated in a culture medium, comprising: an imaging means for imaging the culture medium in which the colonies have occurred; and a connection corresponding to the colonies based on image data obtained from the imaging means. Means for obtaining an area, for each of the obtained connected areas, a means for obtaining the ratio of the long side to the short side of the circumscribed rectangle circumscribing the connected area and having the longest side, Means for integrating a weighting factor that is predetermined according to the ratio and that corresponds to the ratio obtained for each of the connected regions. 2. A colony counting device for counting the number of colonies generated in a culture medium, comprising: imaging means for imaging the culture medium in which the colonies have occurred; and connection corresponding to the colonies based on image data obtained from the imaging means. Means for obtaining an area; for each of the obtained connected areas, a normal to a tangent at each point on the outline of the connected area is obtained, and the intersections are concentrated according to the distribution of the intersections of the normals. A colony counting device, comprising: means for calculating the number of locations where there are; and means for integrating the number obtained for each of the connected regions. 3. The means for determining a connected area corresponding to a colony includes means for determining a connected area having a density corresponding to a colony based on image data obtained from the imaging means. The colony counting device according to claim 1 or 2, wherein the connected region is a connected region corresponding to a colony. 4. The means for determining a connected area corresponding to a colony includes means for determining a connected area having a density corresponding to a colony based on image data obtained from the imaging means, and a density corresponding to a colony. Means for determining the size of the connected region, including, the connecting region having a size equal to or greater than a predetermined size among the connected regions having a concentration corresponding to the colony, as a connected region corresponding to the colony, The colony counting device according to claim 1 or 2, wherein 5. The means for determining a connected region corresponding to a colony includes an extracting means for extracting a contour line corresponding to a colony based on image data obtained from the imaging means, wherein the extracting means comprises a density gradient. The colony counting device according to claim 1, wherein a position of a peak value where the peak value falls within a predetermined range is obtained, and the contour is obtained based on the position. 6. The apparatus according to claim 1, wherein said means for obtaining a connected area corresponding to a colony includes means for masking an area outside the colony counting range based on image data obtained from said imaging means. 6. The colony counting device according to any one of 1 to 5. 7. The colony counting device according to claim 1, wherein said imaging means is a scanner having a one-dimensional image sensor and scanning said one-dimensional image sensor. 8. The storage device according to claim 1, further comprising a storage unit configured to store the image data obtained from the imaging unit and the integrated value obtained by the integration unit in association with each other. A colony counting device according to any one of the above. 9. A medium in which a colony has occurred is imaged by an imaging means, and a connected region corresponding to the colony is determined based on image data obtained from the imaging means, and the number of colonies indicated by one of the obtained connected regions is determined. A ratio of a long side to a short side of a circumscribed rectangle circumscribing the connected area and the longest side being the longest with respect to the obtained one connected area. It is a weighting factor predetermined according to the ratio, a weighting factor corresponding to the ratio determined for the connected region, the number of colonies indicated by the connected region, Identification method to be used. 10. A medium in which a colony has occurred is imaged by an imaging unit, and a connected region corresponding to the colony is determined based on image data obtained from the imaging unit. The number of colonies indicated by one of the determined connected regions An identification method for identifying a tangent at each point on the outline of the connected region with respect to the determined one connected region, An identification method, wherein the number of locations where intersections are concentrated is determined, and the number is determined as the number of colonies indicated by the connection region.